Tissue culture...

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linton

 
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Tissue culture...

by linton » Tue Nov 18, 2008 9:39 am

Does anyone know what the processes are when it comes to growing plants under lab conditions from small pieces of plant tissue (tissue culture)?
1. What are the optimum conditions for success?
2. What is used to ensure that the tissue and growing container are sterilised?
3. What is the growing media (agar I'm guessing) and what is added to it to help the tissue grow?
4. Can it be done at home or do I need specialist equipment or a lab?

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Re: Tissue culture...

by Matt » Tue Nov 18, 2008 2:43 pm

Linton,

I'm trying my hand at tissue culture for the first time today. I'm going to start with seeds. I'll probably try some plant tissue also, but this time of year choosing good tissue is tough because my plants are mostly dormant, so there isn't much new growth. Choosing new growth to work with is important because the older the tissue, the more overrun with bacteria it is, which means it's harder to sterilize. Let me see if I can answer your questions:
linton wrote:1. What are the optimum conditions for success?

This is a hard question to address because there are so many conditions, but the basic answer is sterile media and sterile tissue (seeds, flower stalk or plant tissue).
linton wrote:2. What is used to ensure that the tissue and growing container are sterilised?

To sterilize the growing container and media most people that do TC at home use a pressure cooker. I have an 8 quart pressure cooker. Most pressure cookers are rated at 15PSI, which is ideal for sterilization. You fill the growing containers (most people use baby food jars) with the media and sterilize at 15PSI for 20 to 30 minutes (time depends on the volume of media in each container and what elevation you're at).

Sterilizing the tissue is the challenging part. For Dionaea, seeds are the easiest to sterilize, followed by flower stalk, followed by cuttings. The sterilization techniques vary, but here's what I'm going to try today:
For seeds:
1) 10% bleach for 15 minutes
2) Rinse in sterile water
3) Transfer to media

For tissue:
1) Dip the cutting in the 90% alcohol quickly. Hold it above the alcohol to let any excess drain.
2) Drop the cutting in 3% peroxide for 2 minutes
3) Drop the cutting in 10% bleach mix for 15 minutes
4) Drop the cutting in sterile water for 10 minutes
5) Drop the cutting in 2% PPM (plant preservative mixture) solution for 30 to 60 minutes
6) Transfer to media

linton wrote:3. What is the growing media (agar I'm guessing) and what is added to it to help the tissue grow?

I think this is a common misconception. Agar is inert and doesn't provide anything to the media other than to help it solidify a bit so that the seeds or tissue isn't floating around. The media for carnivorous plants is typically 25% to 50% (I'm going to try 50%) concentration of Murashige & Skoog (MS) basal salt mixture. To stimulate root growth, Auxins are used. And to stimulate cell growth, Cytokinins are used. For an Auxin, I think most people use IBA and for a Cytokinin, BAP is commonly used. I've read varying ratios, but 5ml BAP to 0.25ml IBA per liter of media is what one person that TCs Dionaea told me he uses. The TC kit that I have recommends 3ml of BAP per liter of media. From what I've read, for Dionaea you don't even really need it. I think I'm going to try some with and some without it. The other thing that is added to the media is sugar. Regular table sugar is fine and it should be 2 tablespoons (30g) per liter of media.

linton wrote:4. Can it be done at home or do I need specialist equipment or a lab?

It can be done at home. I ordered my kit from here:
http://www.kitchenculturekit.com/index.htm
It comes with a manual and it is a very good place to start. I'd love it if someone else on this forum got into TC and we could exchange notes. It sounds like sterilizing tissue is a trial and error process and I can't seem to get many people that have been successful to share their technique with me. So, it would be very valuable to have another person working on TC at the same time so that we could exchange notes.
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Re: Tissue culture...

by linton » Wed Nov 19, 2008 8:39 am

Thanks Matt, this answers a lot of my questions and raises some more too - there are a lot of things (mixtures / additives / hormones etc) that I have not heard of before involved in the process. I think that without a book or manual, I'd be completely lost after sterilising the tissue!! But I might just start looking around for a second hand pressure cooker.
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Re: Tissue culture...

by lemonlily » Tue Dec 09, 2008 2:44 am

What part of the plant is tissue? (I don't mean tissue you sneeze on but you know what I mean).
Would it hurt a plant if you take tissue out of it? (If thats what you do)
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Re: Tissue culture...

by Matt » Tue Dec 09, 2008 4:33 am

lemonlily wrote:What part of the plant is tissue? (I don't mean tissue you sneeze on but you know what I mean).
Would it hurt a plant if you take tissue out of it? (If thats what you do)

You can use the leaves, traps, roots or flower stalk for tissue. But the dirtier the tissue is, the hard it is to sterilize. Typically flower stalks are fairly easy because they don't contact the soil. Traps and leaves would be next in order of difficulty to sterilize and finally the roots.
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Re: Tissue culture...

by lemonlily » Tue Dec 09, 2008 5:55 am

Matt wrote:
lemonlily wrote:What part of the plant is tissue? (I don't mean tissue you sneeze on but you know what I mean).
Would it hurt a plant if you take tissue out of it? (If thats what you do)

You can use the leaves, traps, roots or flower stalk for tissue. But the dirtier the tissue is, the hard it is to sterilize. Typically flower stalks are fairly easy because they don't contact the soil. Traps and leaves would be next in order of difficulty to sterilize and finally the roots.


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Re: Tissue culture...

by Matt » Tue Dec 09, 2008 7:01 am

Because soil has a lot of bacteria in it.
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Re: Tissue culture...

by lemonlily » Sun Feb 15, 2009 12:51 am

If soil have a lot of bacteria, why do plants live with it? I know that everyting and everywhere has bacteria, but it is interesting that it doesn't do anything, really.
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Re: Tissue culture...

by Matt » Sun Feb 15, 2009 2:11 am

lemonlily wrote:If soil have a lot of bacteria, why do plants live with it? I know that everyting and everywhere has bacteria, but it is interesting that it doesn't do anything, really.

Because the plants have no choice but to live with it. When you put them in an environment where there isn't any bacteria, they grow much faster, so it does stuff for sure. It's just that everything has adapted to living with it, so it looks like it doesn't do anything.
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Re: Tissue culture...

by lemonlily » Sun Feb 15, 2009 3:08 am

I've never thought of it that way, I wonder if I don't have bacteria on me, would I grow faster? :lol: Seems pretty impossible to figure out as nothing is bacteria free, but does anyone have a guess?
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Re: Tissue culture...

by WORMSS » Sun Feb 15, 2009 10:25 am

Onion: We could try putting You in a pressure cooker for 15 minutes if you like?
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Re: Tissue culture...

by lemonlily » Sun Feb 15, 2009 8:13 pm

Oh yeah, sure. Lets see you jam me in there. I think since you are not as clean as I am so you should try first. It would be nice to have human meat for dinner!
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Re: Tissue culture...

by NZL » Fri Apr 24, 2009 6:53 am

Lily,

Your intestines are filled with bacteria! But they are symbiotic. They help our body in a few ways. For example: they help digest our food so our body wastes less, and they also eat a little bit of our food to produce vitamins (especially vitamin K). Now, the definition of a vitamin includes that it is something that is vital for us to eat, because our body can't produce it on its own.
Other examples of bacteria you may have encountered before is when you're sweating. Sweat doesn't smell. It's the bacteria on your skin that make the smelly stuff (body odor) and when you sweat, this smell is released much easier.
Most bacteria are harmless but there are some little nasty buggers out there too.
Meningitis, laryngitis, ulcer, skin rash etc can all be caused by bacteria.
But the most dangerous bacteria out there at this moment are the MRSA (methicillin-resistent Straphylococcus aureus)
These bacteria are immune to all but one class of antibiotics. When they become resistent to this last class, mankind will not have any means to combat these bacteria, which can lead to a pandemic infection.
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Re: Tissue culture...

by hackerberry » Fri Apr 24, 2009 4:56 pm

Guys, we're moving out of the main topic here.

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Re: Tissue culture...

by NZL » Fri Apr 24, 2009 6:03 pm

I know, I'm sorry.
I just wanted to bump the topic for future reference. The mailman delivered my TC kit today, but i wasn't home, so i have to go to the post office next week ;)

This all happened before I learned how to bookmark a topic :oops:
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